The present invention is related to a nucleic acid construct comprising an expression unit for the expression of E1A and an expression unit for the expression of E1B, a vector comprising such nucleic acid construct, a cell comprising the nucleic acid construct and/or the vector, a method for the production of a permanent amniocytic cell line comprising the step of introducing the nucleic acid construct and/or the vector, a permanent amniocyic cell line, the use of the cell, a method for the production of a gene transfer vector or an adenovirus mutant, and a method for the production of a protein.
1. Adenovirus and Adenovirus Infectious Cycle
Adenoviruses are non-enveloped viruses belonging to the virus family Adenoviridae. They carry a linear double-stranded DNA genome with a size of about 36 kilobases (kb). The viral genome contains at both ends the inverted terminal repeat sequences (ITRs) as origin of replication and at the left end a packaging signal. Adenoviruses have been isolated from many vertebrate species including humans and chimpanzees. More than 50 human serotypes can be distinguished based on DNA sequence. During an infectious cycle the viral particle enters the cell by receptor-mediated endocytosis and the viral genome enters the nucleus as DNA-protein complex. The adenoviral infection cycle is divided into an early and a late phase, which are separated by the start of adenoviral replication (Shenk, in: Virology, Fields ed., Lippincott-Raven Publishing, Philadelphia, pp. 2111-2148, 1996). In the early phase, i.e. before replication, there is expression of the early viral functions E1, E2, E3 and E4. The late phase is characterized by transcription of late genes, which are responsible for the expression of viral structural proteins and for the production of new viral particles.
E1A is the first viral gene expressed after the viral genome enters the nucleus. The E1A gene codes for the 12S and 13S proteins, which are formed by alternative splicing of the E1A RNA. By binding to several cellular proteins including pRB, p107, p130, p300 (CBP), p400, TRAP and others (Berk, 2005), the E1A proteins activate cellular DNA synthesis, promote S-phase entry, activate and repress, respectively, a large number of cellular genes, thereby instructing the cell to allow a viral infectious cycle. In addition, E1A activates most other adenovirus genes including E1B, E2, E3, E4 and the major late transcription unit (MLTU). Expression of E1A on its own leads to apoptosis.
E1B is one of the early viral genes activated by E1A. The E1B gene codes for several proteins, including the well-known E1B 55 kD and E1B 19 kD proteins, which are generated by alternative splicing of the E1B RNA. The E1B 55 kD (also called E1B 55K) protein modulates the progression of the cell cycle by interacting with the p53 tumor suppressor, is involved in preventing the transport of cellular mRNA in the late phase of the infection, and prevents E1A-induced apoptosis of cells. The E1B 19 kD (also called E1B 19K) protein is likewise important for preventing E1A-induced apoptosis of cells.
Rodent cells can be easily transformed in cell culture by expression of the E1A and E1B proteins and in rodent cells co-expression of the E1A and E1B proteins is considered to be necessary and sufficient for the transformation event to occur. In addition to transcripts coding for the E1B 55K and 19K proteins, three further E1B transcripts, also generated by alternative splicing have been identified (E1B-156R, E1B-93R and E1B-84R), one of which (E1B-156R) has been shown to promote transformation (Sieber et al. 2007). In the context of the wildtype adenoviral genome, all E1B transcripts use a common downstream splice acceptor that overlaps with part of the 5′-untranslated transcript of the pIX gene (i.e. between the pIX promoter and the translational start of pIX). In hAd5 (NCBI Reference Sequence: AC_000008) this splice acceptor is located at nucleotide 3595 of the hAd5 genomic sequence.
The next genes to be expressed during an infectious cycle are the E2A and E2B genes coding for three proteins (preterminal protein, pTP; DNA Polymerase, Pol; and DNA-binding protein, DBP), all involved in replication of the viral genome.
E3 is mainly involved in counteracting host defenses against adenoviral infection and is dispensable for virus grows in cell culture.
E4, also expressed early in an infectious cycle, codes for various proteins. In addition to other functions E4 blocks, together with the E1B 55K protein, the accumulation of cellular mRNAs in the cytoplasm, and at the same time it facilitates the transport of viral RNAs from the cell nucleus into the cytoplasm.
The initiation of DNA replication is followed by expression of structural proteins, which are necessary for the formation of the viral capsid and for condensation of the viral DNA. Late during an infectious cycle the viral DNA is packaged into the viral capsid. The exact mechanism of the packaging of the viral genome into the viral capsid is currently unknown, but involves interaction of several virus-encoded proteins with the packaging signal located at the left terminus of the viral genome.
2. Adenovirus Vectors
Different vector types based on adenovirus have been developed (McConnell et al. 2004; Imperiale et al. 2004).
Adenoviral vectors usually have at least deletions of the E1A and E1B genes and are therefore replication-deficient in human cells. Production takes place in human complementing cell lines, which express the E1A and E1B proteins and in which the E1A and E1B genes are chromosomally integrated.
The ΔE1Ad vector (also called E1-deleted Ad vector or first-generation Ad vector) is the dominant vector type, which is widely used as laboratory tool, in pre-clinical R&D, in clinical studies and product development in the context of gene therapy or genetic vaccination. This vector type is made replication-defective in primary cells by removal of the E1 region (ΔE1) encoding the E1A and E1B proteins.
Many ΔE1Ad vectors also contain partial or complete deletion of the E3 region (ΔE1/ΔE3 Ad vectors), since E3, among other functions modulating virus-host interaction and interfering with the immune system, is dispensable for vector production in cell culture. So far, most ΔE1Ad vectors are based on human adenovirus type 5 (hAd5). However, vectors based on other human (e.g. hAd6, hAd26, hAd35 and others) and non-human adenovirus types (e.g. derived from Chimpanzee) have been developed (Bangari et al. Vacci 2006).
Second-generation vectors are based on ΔE1Ad vectors that carry additional mutations in other early regions of the viral genome, including the E2 genes and/or the E4 genes (Imperiale et al. Curr Top Microbiol Immunol 2004, 273, 335-57). They are produced in cell lines, in which, in addition to the E1A and E1B genes, also the respective adenoviral gene or genes that is/are mutated in the vector's genome are expressed. For example, Ad vectors with deletion of the DNA binding protein (DBP) that is one of the E2 genes are produced in cell lines, which express the DBP in addition to the E1A and E1B genes.
In high-capacity Ad (HC-Ad) vectors (also called helper-dependent Ad vectors) all viral coding sequences are replaced by the transgene(s) of interest. In most cases additional stuffer DNA are included in the vector to prevent rearrangements during production. Current production systems are based on the use of a replication-deficient (ΔE1) helper virus providing all non-structural and structural viral functions in trans together with a production cell line expressing either Cre or Flp recombinase (Parks et al., 1996; Umana et al., 2001).
Production and purification methods of Ad vectors in adherent or in suspension cell culture are well known to the expert and have been described (Silva et al., 2010).
3. Generation of Producer Cell Lines by Transformation of Human Cells with the E1A and E1B Genes
Traditionally, ΔE1Ad vectors have mainly been produced in 293 cells, which were generated by transfection of human embryonic kidney (HEK) cells with sheared DNA of human Adenovirus type 5 (Graham et al., 1977). In a total of eight transfection experiments, with an average of twenty HEK cultures used per experiment, only a single immortalized cell clone was obtained (Graham et al., 1977). HEK 293, the cell line established from this cell clone, contains chromosomally integrated nucleotides (nt.) 1 to 4344 of the Ad5 genome, including the E1A and E1B genes, left ITR and the adenoviral packaging signal (Louis et al., 1997).
Although rodent cells can easily be transformed with adenoviral E1 functions, primary human cells have been found to be notoriously difficult to transform with the E1A and E1B genes. Gallimore and coworkers attempted to transform primary HEK cells with E1 functions of Ad12 (Gallimore et al., 1986). These experiments were carried out unsuccessfully over a period of three years with more than 1 mg of the EcoRI cDNA fragment of Ad12, containing the E1A and E1B genes. Despite a large number of experiments carried out, only four Ad12-E1 HEK cell lines were isolated (Whittaker et al., 1984). Likewise, the same group failed to transform other primary human cells with E1 functions, including keratinocytes, skin fibroblasts, hepatocytes and urothelial cells (Gallimore et al., 1986). One cell type reproducibly transformed with adenoviral E1 functions are human embryonic retinal cells (HER cells) (Byrd et al., Nature 298, 69-71, 1982). Although the transformation efficiency of HER cells was lower than that of primary rat cells, it was more than 100 times higher than that of HEK cells. The investigations were initiated in order to produce complementing cell lines for the isolation of Ad12 E1 mutants.
Transfection of HERs with a construct containing an hAd5 fragment from nt 79 to 5789 resulted in a cell line, named 911, which supported the growth of ΔE1Ad vectors and at least matched production yield of 293 cells (Fallaux et al., 1996). However, due to extensive overlap with ΔE1Ad vectors both 911 and 293 cells are prone to the regular generation of replication competent adenovirus (RCA) as a result of homologous recombination events between the vector genome and the chromosomally integrated E1 region during production (Lochmüller et al., 1994; Hehir et al., 1996). Importantly, this is a frequent occurrence that can neither be controlled nor avoided in particular during serial passage of vectors and large-scale vector production. The U.S. Food and Drug Administration (FDA) guidelines demand the presence of less than one RCA in 3×1010 vector particles for clinical applications (Biological Response Modifiers Advisory Committee, 2001).
To circumvent and/or prevent the risk of RCA emergence during Ad vector production, other E1-transcomplementing cell lines harbouring a minimized E1 DNA fragment lacking any homology with the DNA of commonly used Ad vectors have been developed. In particular, HER cells were transformed with a new E1A and E1B encoding construct, in which any identical sequences/sequence overlap with ΔE1Ad vectors were/was eliminated. By replacing the E1A promoter by the human phosphoglycerate kinase (PGK) promoter and the 3′-untranslated region (3′UTR) of E1B by the mRNA processing elements of the hepatitis B virus surface (HbS) antigen (not containing an intron), the E1-transformed cell line PER.C6 was generated solely encompassing hAd5 sequences from nt. 459 to 3510 (Fallaux et al., 1998). Accordingly, matching ΔE1Ad vectors lacking this region can be efficiently propagated in these cells without the occurrence of RCA due to homologous recombination. However, in two publications about PER.C6 cells unusual vector recombinants have been observed, that result in vector specimen carrying and expressing E1 functions. In the first report (Murakami et al., 2002), in which the vector did have an overlap of 177 nt. with the integrated E1 region, helper-dependent E1-positive particles (HDEPs) were generated caused by one homologous and one heterologous recombination event, resulting in the concomitant deletion of parts of the adenoviral vector backbone. As a result the Ad vector preparation contained two different particle species: the original ΔE1 vector and the E1 region-containing recombinant. In a second report (Murakami et al., 2004) E1 region-positive recombinant particles were described although the parental vector sequence did not overlap with the integrated E1 region. Detailed analysis of several different independent E1-positive isolates showed a similar structure of recombinants, consisting of a palindromic structure of several copies of the E1 region flanked by the adenoviral left ITR including the packaging signal. According to the authors' interpretation, the recombinants most likely were generated following heterologous recombination between the ΔE1Ad vector and the chromosomal DNA close to the E1-region. The authors further speculate, that the generation of the E1-positive recombinants is facilitated by the observed head-to-head dimer structure of (some of) the 10 to 20 E1 region integrates that are present in PER.C6 cells.
Some non-hAd5 based ΔE1Ad vectors, an example being vectors based on hAd35, cannot be propagated in regular production systems such as 293 cells or PER.C6 cells, since both express E1A and E1B of hAd5, while hAd35-based vectors require for their production E1B functions of hAd35. Thus, for production of such vectors, the missing function needs to be provided in the production cell line. In case of hAd35-based vectors, for example, an E1B function of hAd35 has to be provided by the cell line (Vogels et al., 2003, Gao et al., 2003).
More recently, human amniocytes were identified as an alternative cell source for the generation of cell lines following transformation with E1 functions (Schieder et al., 2000) and E1/pIX genes (Schieder et al., 2008). The design of the E1A and E1B expressing plasmid construct in the cell line N52.E6 (Schiedner et al., 2000), was similar as in PER.C6 cells, in principle excluding the generation of RCA during vector production due to the absence of any sequence overlap between vector DNA and the integrated E1 region.
There have been additional attempts to generate production cell lines for ΔE1 vectors. Unlike the cell lines discussed above, they all were based on established cell lines such as HeLa and A549 cells although, due to the poorly documented generation of the original cell lines, their tumorigenic origin and their high tumorigenicity, they are not suitable for production of clinical grade material (reviewed in Silva et al., 2010).
4. Immortalization of Primary Cells in Cell Culture
Mammalian cells, when isolated from an animal or a human, taken into a cell culture dish and provided with proper nutrients, can be cultured by serial passaging only for a limited time. This phenomenon has been first described by Hayflick (Hayflick and Moorhead, 1961) and is called cellular senescence. Senescent cells in cell culture undergo changes in their morphology and become large and flattened; they stop dividing while remaining metabolically active. There are distinct changes in gene expression, protein processing and metabolism and, as useful marker, cells stain positive for senescence-associated β-galactosidase (SA-β-gal) (Weinberg, R. A., The Biology of Cancer, 2007, Garland Science). The limitation in replicative potential of primary mammalian cells in cell culture and senescence is mainly associated both with cell-physiologic stress factors due to cell culture conditions (characterized by alteration in specific signaling pathways, such as frequent upregulation of p16/INK4a and others (Ben-Porath and Weinberg, 2005)) and with reduction of telomere length at the chromosomal ends due to the so-called endreplication problem that occurs during replication of cellular DNA (Weinberg, R. A., 2007 supra). Telomeres are structures located at the end of chromosomes, consisting of short hexanucleotide DNA repeats and being associated with a number of proteins, protecting the integrity of chromosomes and preventing, for example, fusion events between different chromosomes. Telomere length is maintained by the activity of several proteins including the essential telomerase holoenzyme that consists of the catalytic subunit hTERT and an RNA subunit (hTR). In primary cells, the activity of hTERT is too low to maintain telomere length constant, resulting in a gradual loss of telomeric repeats during replication of the cellular DNA. In humans, the number of replicative doublings a primary cell can maximally go through before entering senescence is ranging from about 50 to 60 population doublings (PD), slowly decreasing when cells are isolated from individuals with increasing age (Weinberg, R. A., 2007 supra). The number of PDs is also dependent on the specific cell type and the cell culture conditions. Some cells can be taken into cell culture only for a few PDs, other for a larger number, however not beyond far the limit mentioned above.
Cells, that can be maintained in cell culture indefinitely, when they are provided with appropriate nutrients, are said to be immortal and such cells can also be called a cell line or a permanent cell line. Primary normal human cells usually do not become immortalized spontaneously. However, immortalization can be achieved experimentally, for example by introducing cellular or viral oncogenes or by introducing mutations in tumour suppressor genes.
Crisis is a term that is mechanistically linked to the reduction of telomere length to a point that most cells will undergo cell death. This can be observed, for example, when tumour cells are taken into cell culture. After a certain number of replicative doublings, most of the cells will undergo cell death due to telomere length shortening. Only rarely individual cells will survive, generally selected for increased growth rate and survival by additional mutations. When primary human cells, for example human fibroblasts or epithelial cells are taken into cell culture, it is frequently observed that cells can be maintained by passaging for a small number of PDs until they acquire a senescent phenotype. This early type of senescence can be delayed, for example by the expression in these cells of the large T Antigen of SV40 (Weinberg, R. A., 2007 supra), resulting in the inactivation of the oncoproteins pRB and p53. However, after a certain number of PDs and depending on the length of the remaining telomeres the cells will enter crisis due to telomere collapse. Only cells, which manage to either activate telomerase or to engage an alternative way of telomere maintenance—called alternate lengthening of telomeres (ALT)—have a chance to survive. According to current understanding, crisis is the time, when structural abnormalities of the karyotype are preferentially established, due to fusion events between eroded (telomere depleted) chromosomal ends, followed by so-called breakage-fusion-bridge (BFB) cycles, resulting in karyotypic chaos (Weinberg, R. A., 2007 supra). These abnormalities, in combination with other mutations occurring during culture, furnish some cells with a selective growth advantage, enabling them to evade from crisis and become immortalized.
5. Use of Human Cells for the Production of Biologics
Human cells are of significant interest to the industry for the production of certain biologics such as viral vectors, proteins, viruses and vaccines for therapeutic, diagnostic or prophylactic human or veterinary use. Examples for viral vectors that can be used for therapeutic or prophylactic purposes are vectors that are based on different viruses including adenovirus, retrovirus, herpes simplex virus or parvovirus. Most of viral vectors used today are produced in human cell lines such as 293 cells. They can be used either for functional studies, for therapeutic purposes such as gene therapy or for therapeutic or prophylactic purposes such as genetic vaccination. Proteins that cannot be produced in simple organism such as bacteria or that are characterized by certain posttranslational modifications frequently require the use of mammalian cells for their production. Examples of biologics that can be produced in human cells are therapeutic or diagnostic antibodies or therapeutic glycoproteins including for example blood coagulation factors or fibrinolytic proteins. Many human vaccines are based on inactivated or attenuated human viruses that grow well on human cells. Also many subunit protein vaccines or complex vaccines such as virus-like-particles (VLPs) can be produced in human cells.
For production of biologics at an industrial scale, however, the use of permanent cell lines rather than of primary cells is a necessity. In general, primary cells can often not be expanded to a sufficient amount to allow production of proteins or viruses at a scale large enough for market supply. While permanent cell lines can be grown to a very large cell number, either as adherent cell culture or in suspension, it is well known, that genetic stability of cultured cells is difficult to maintain, for example due to telomere shortening during the process of immortalization or due to oxidative stress during cell culture resulting in mutations. However, genetic stability of cell lines is very important for the industrial production of well-characterized products of high quality (e.g. characterized by consistent glycosylation of glycoproteins), activity (e.g. characterized by consistent immunogenicity of vaccines) and uniformity (e.g. little variation of the product between different production runs).
Thus, the problem underlying the present invention is to provide means which allow the generation of a genetically stable cell line.
A further problem underlying the present invention is to provide a genetically stable cell line.
A still further problem underlying the present invention is to provide means which allow the practicing of a method for the improved generation of immortalized and genetically stable human cell lines which may, among others, be used in the production of therapeutic, diagnostic or prophylactic biologics for human or veterinary use.